Application of stiff stiff for preparing medicine for inhibiting angiogenesis

Abstract

The invention provides an application of stiff stiff (Beauverin) in preparation of a medicine for inhibiting angiogenesis, wherein the effective treatment concentration range of the stiff stiff is 0.1 mu M to 5 mu M. The invention further provides an application of the stiff stiff in the preparation of the medicine for inhibiting the angiogenesis. The compound can be used for preparing a medicine for treating or preventing retinopathy, or can be combined with an anti-cancer medicine to form a medicine composition or a medicine combination for treating cancers.

Description

Use of chrysogenum styracifluanicum for preparing angiogenesis-inhibiting drugs Technical Field

[0001] The present invention provides a use of styraclonitin for preparing a drug for inhibiting angiogenesis, which can be used in the development of drugs for treating or preventing retinopathy, and pharmaceutical compositions or combinations for treating cancer. Background Art

[0002] Cancer develops due to abnormal cell hypertrophy and proliferation, which metastasize and invade other tissues through the body's circulatory and lymphatic systems, causing organ dysfunction and failure, leading to disease. Cancer cells have been shown to be closely linked to angiogenesis, a process by which pre-exiting vessels sprout to form new blood vessels. Tumor cells require large quantities of nutrients and oxygen for growth and spread, which are supplied by blood vessels. Tumors often promote angiogenesis in surrounding tissues, forming a larger vascular network to meet their growth and metabolic needs. Therefore, inhibiting angiogenesis is significantly effective in controlling the onset of cancer.

[0003] Angiogenesis is also related to the occurrence of related eye diseases. Poor blood circulation in the eyes often leads to hypoxia of retinal tissue and the secretion of vascular endothelial growth factor (VEGF). VEGF should play a compensatory role in angiogenesis to combat the hypoxic environment. However, in most pathological conditions, these newly formed blood vessels are thin and dense, not only failing to increase blood supply, but also increasing the permeability and leakage of the blood vessel walls. The accumulated fluid further hinders blood flow, forming a vicious cycle.

[0004] Previous inventions have disclosed a beauvericin compound and a pharmaceutical composition thereof, which have the potential to improve eye diseases caused by angiogenesis, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR).

[0005] However, drugs that inhibit angiogenesis for the treatment or prevention of retinopathy or the treatment of cancer have yet to be developed.

[0006] Summary of the Invention

[0007] Therefore, the present invention provides a compound for use in preparing a medicament for inhibiting angiogenesis, wherein the compound is beauvericin having the following structure, referred to herein as GYT088, and the therapeutically effective concentration range of beauvericin is 0.1 μM to 5 μM:

[0008] According to an embodiment of the present invention, the angiogenesis-inhibiting drug can be developed as a drug for treating or preventing retinopathy.

[0009] According to an embodiment of the present invention, the retinopathy is age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.

[0010] In one embodiment of the present invention, the angiogenesis-inhibiting drug can be developed as a drug for treating or preventing retinopathy.

[0011] According to an embodiment of the present invention, the retinopathy is age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.

[0012] According to an embodiment of the present invention, for humans, the therapeutically effective concentration of the styraxine is preferably in the range of 0.5 μM to 5 μM.

[0013] According to an embodiment of the present invention, the dosage range of the sclerotin for treating or preventing retinopathy is 2.0 μg to 5.5 μg per eye.

[0014] In another aspect, the present invention provides a pharmaceutical composition for inhibiting angiogenesis, comprising a therapeutically effective concentration of chlamydomonas aeruginosa in the range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier.

[0015] According to an embodiment of the present invention, the therapeutically effective concentration of the styraxine is preferably in the range of 0.5 μM to 5 μM.

[0016] According to an embodiment of the present invention, the pharmaceutical composition can be combined with an anticancer drug to prepare a pharmaceutical composition or pharmaceutical combination for treating cancer.

[0017] It is to be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing summary of the invention and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the accompanying drawings presently preferred embodiments.

[0019] Figure 1 shows a schematic diagram of the test process time and process.

[0020] Figure 2 shows the imaging results and data analysis of fundus photography (FP) and fluorescein fundus angiography (FFA): Figure 2(A) shows the results on day 0 after laser injury; Figure 2(B) shows the results on day 7 after laser injury; and Figure 2(C) shows the results on day 14 after laser injury. Figure 2(D) shows the quantitative results of fluorescein fundus angiography. * p<0.05 indicated a significant difference between the high-dose GYT088-treated group (5.12 ng / eye) and the other groups on day 7. ** p<0.01 indicated significant differences between the Eylea-treated group and the other groups on day 7. ## p<0.01 indicates that the medium-dose and high-dose GYT088-treated groups (2.56 and 5.12 ng / eye) on day 14 were significantly different from the other groups. ### p<0.001 showed that the Eylea-treated group on day 14 was significantly different from the other groups.

[0021] Figure 3 shows the imaging results of spectral domain optical coherence tomography (SD-OCT) at the laser-induced injury site and the analysis of retinal sublayer structure data: Figure 3(A) shows the results on day 0 after laser injury; Figure 3(B) shows the results on day 7 after laser injury; and Figure 3(C) shows the results on day 14 after laser injury. Figure 3(D) shows the quantitative assessment of retinal structural lesions observed using SD-OCT images. * p<0.05 indicated significant differences between the Eylea-treated group and the other groups on day 7. ### p<0.001 showed that on day 14, there were significant differences between the Eylea, low-dose, medium-dose and high-dose GYT088 treatment groups (1.28, 2.56, 5.12 ng / eye) and the control group.

[0022] The details of one or more embodiments of the present invention are set forth in the following description. Other features and advantages of the present invention will become apparent from the following detailed description of several specific embodiments and the appended claims. DETAILED DESCRIPTION

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs.

[0024] The present invention is further described by the following examples, which are provided for illustration and not for limitation.

[0025] As used herein, the article "a" or "an" refers to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0026] The term "angiogenesis," as used herein, refers to the process of new blood vessel formation. Vascular endothelial growth factor (VEGF) promotes the proliferation and migration of endothelial cells, leading to tissue growth, healing, or repair. This physiological process is crucial for normal development and maintenance of tissue function. However, when angiogenesis becomes unbalanced, it can contribute to disease, particularly in conditions such as cancer and chronic inflammation.

[0027] The term "cancer," as used in this article, refers to a disease caused by the uncontrolled growth and division of a group of abnormal cells. These abnormal cells are called tumors, and cancer generally refers to malignant tumors that continuously divide and circulate throughout the body, invading other normal cells and tissues, causing malfunction or organ failure.

[0028] The term "retinopathy," as used in this article, refers to various diseases that affect the retina. Because the retina, the tissue inside the eye, receives light and converts it into nerve signals for transmission to the brain for visual perception, retinal damage or disease can lead to impaired or lost vision. Common retinopathy conditions include age-related macular degeneration (AMD), diabetic retinopathy, glaucomatous optic neuropathy, retinal detachment, and retinal vascular diseases.

[0029] As used herein, the term "therapeutically effective concentration" refers to a concentration of a compound or agent that results in the treatment, cure, prevention, or amelioration of a disease, condition, or side effect, or a reduction in the rate of progression of a disease or condition, compared to a corresponding individual not receiving that concentration. The term also includes within its scope concentrations effective to enhance normal physiological function.

[0030] The term "Beauvericin" as used herein refers to compounds produced as secondary metabolites by fungi of the genus Beauveria. It belongs to a class of compounds known as cyclic peptides and has a unique structure as follows:

[0031] The structural characteristics of schizolin include an alternating arrangement of three D-hydroxyvaleric acids and three N-methyl-phenylalanine amino acids. Schizolin has diverse biological activities, including antibacterial, insecticidal, and anticancer activities against various tumor cell lines.

[0032] The present invention discovered that styraxine has the effect of inhibiting angiogenesis at a specific low dose. It can be used to develop drugs for preventing or treating retinopathy, or to prepare a pharmaceutical composition or combination for treating cancer in combination with an anticancer drug.

[0033] According to embodiments of the present invention, based on dosage conversion in mice, the effective therapeutic dose of sclerotin is in the range of 2.56 ng to 5.12 ng per eye. Therefore, for humans, the dose range of sclerotin for treating or preventing retinopathy is 2.0 μg to 5.5 μg per eye. It can be estimated that the effective concentration range of sclerotin for inhibiting angiogenesis is 0.1 μM to 5 μM, preferably 0.5 μM to 5 μM.

[0034] For treatment, the compound at a therapeutically effective concentration is formulated into a pharmaceutical composition for administration. Therefore, the present invention further provides a pharmaceutical composition comprising a therapeutically effective concentration of styraxine in the range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier.

[0035] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier, diluent, or excipient that is acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject to which the pharmaceutical composition is administered. Depending on the requirements of the pharmaceutical formulation, any carrier, diluent, or excipient commonly known or used in the art may be used in the present invention.

[0036] In the present invention, the pharmaceutical composition can be administered by any suitable route, including but not limited to oral, rectal, nasal, topical, vaginal, or parenteral routes. In a specific embodiment of the present invention, the pharmaceutical composition is formulated for oral administration or injection. Such formulations can be prepared by any method known in the pharmaceutical art.

[0037] The present invention is further described with reference to the following examples, which are not intended to limit the scope of the present invention.

[0038] Example Trial on the Effect of Mycobacterium serratum on the Improvement of Retinopathy

[0039] 1. Test materials

[0040] Beauvericin (BEA), hereinafter referred to as GYT088, was provided by Ziyuantang Biotechnology Co., Ltd. 5 mg of BEA (molecular weight 783.96) was dissolved in DMSO to a stock concentration of 50 mM. The solution was then diluted with saline to 100 μM. Before injection, the solution was diluted with saline to form low-, medium-, and high-dose groups with concentrations of 1.6, 3.2, and 6.4 μM, respectively. Based on a mouse vitreous volume of approximately 4 μl, an injection of approximately 1 μl is equivalent to 1.28, 2.56, and 5.12 ng / eye, respectively. Based on a human vitreous volume of 4-4.5 ml, the low-, medium-, and high-dose concentrations are 1.28, 2.56, and 5.12 μg / eye, respectively.

[0041] 2. Experimental Design

[0042] [Corrected 01 / 03 / 2024 according to Rule 26] This experiment used 7-8 week old male C57BL / 6 mice obtained from BioLASCO Taiwan Co., Ltd. (Taiwan, China), weighing approximately 17-20 g. They were acclimated for at least 5 days in the animal room of Taipei Medical University, Taiwan, prior to the start of the experiment. The temperature was set at 23 ± 1°C, the humidity was set at 39-43%, and an automated light control system maintained a 12-hour light and 12-hour dark cycle. Rodent diet 5001 (Purina, MO, USA) was used for feeding, and drinking water was available ad libitum.

[0043] After the adaptation period, the mice were burned with laser excitation light on the retina (day 0). On the same day, fundus photography (FP), fluorescein fundus angiography (FFA), and frequency domain optical coherence tomography (SD-OCT) were used to confirm the extent of retinal damage (Figure 1). The experiment was divided into 5 groups. The mice were randomly divided into groups and given vitreous injections of corresponding drugs, including (1) control group, (2) positive control group given Eylea (40μg / eye / μL), (3-5) experimental groups GYT088 low dose group (1.28ng / eye), medium dose group (2.56ng / eye), and high dose group (5.16ng / eye). There were 5 mice in each group. Clinical observation was performed every day, and the retinal damage repair was examined on the 7th and 14th days.

[0044] 3. Test methods

[0045] 3.1 Laser-induced choroidal neovascularization in mice:

[0046] C57BL / 6 mice were intraperitoneally injected with a dissociative, fast-acting general anesthetic (IP). After deep anesthesia, the mice were placed on a support platform and mydriasis was dilated with atropine. Thirty minutes later, contact gel (OmniVision) was applied. Retinal laser damage was then applied using a double-frequency Nd:YAG laser (Laser LP352; Lumenis Inc., Salt Lake City, UT). Exposures were 0.1 seconds, with a laser energy of 250-300 mW. The lesion area was approximately 100 μm in diameter. Two to four lesions (points) were applied between retinal vessels within the same field of view. Fluorescent leakage areas were recorded on the same day using FFA. Mice were returned to their cages for observation after recovery.

[0047] 3.2 Fundus fluorescein angiography (FFA) and analysis:

[0048] Mice were deeply anesthetized and mydriasis was achieved. Fundus photography (FP) was acquired using a Micron III retinal imaging microscope (Phoenix, San Ramon, CA, USA), with the mouse fovea positioned at the center of the ophthalmoscope. Retinal angiography (FP) was then performed using a 520 nm filter after intraperitoneal injection of a 10% sodium fluorescein solution. Continuous images were acquired and analyzed. Day 0, the day after drug administration, was considered the zero day of the study. FP, FFA, and SD-OCT were observed weekly until day 14. For analysis, clear images were selected and imported into ImageJ in a 16-pixel format. Background was removed, and the software automatically quantified the pixel count and area of ​​the fundus region. Due to individual retinal structural differences, wound repair after laser treatment varies, and there is some signal overexposure in this region. If fluorescence leakage occurs outside the area directly damaged by the laser, it can be considered as a phenomenon of fundus angiogenesis. Therefore, three fixed areas of fluorescence leakage were randomly selected in the non-laser damaged area for quantitative statistics.

[0049] 3.3 Spectral-domain optical coherence tomography (SD-OCT) and analysis

[0050] The OCT (Phoenix Research Laboratories, San Ramon, CA) imaging system used in this study is based on the principle of frequency domain optical coherence tomography. The computed tomography is divided into three layers: the first layer is the Nerve fiber layer (NFL) to the Inner nuclear layer (INL), the second layer is the INL to the Inner segment / outer segment (IS / OS), and the third layer is the IS / OS to the Retinal pigment epithelium (RPE). The degree of damage is scored based on the retinal sublayer structure. The scoring is performed in a double-blind manner, with three people scoring separately and taking the average value as the indicator value of the degree of damage. The scoring criteria are as follows:

[0051] Table 3: Structural damage scores of retinal sublayers.

[0052] 3.4 Statistical methods

[0053] All data are expressed as mean ± standard deviation (SEM) and statistically analyzed by one-way ANOVA (Turkey test), with p < 0.05 considered to be significant.

[0054] 4. Results

[0055] 4.1 Image analysis of retinal fluorescein fundus angiography (FFA)

[0056] [Corrected 12 / 03 / 2024 according to Rule 91] FFA images were analyzed using ImageJ to quantify the area of ​​fluorescence leakage. Since no significant leakage was observed on day 0, the fluorescence was primarily derived from newly formed blood vessels. Images taken on day 0 revealed three laser-induced lesions in the fundus of each group of mice after laser injury. Quantification yielded the relative mean fluorescence leakage ratios for each group on days 0, 7, and 14 after laser injury (Figure 2). Angiogenesis in the GYT088-treated group increased by 1.92±0.21, 1.59±0.22, and 1.53±0.17-fold on day 7, respectively, compared to day 0, with the high-dose group achieving statistically significant inhibition of angiogenesis (p<0.05). On day 14, angiogenesis indicators in the GYT088-treated group increased by 2.46±0.18, 1.79±0.17, and 1.63±0.16 times compared to day 0. The inhibition of angiogenesis in the medium and high-dose groups reached statistical significance (p<0.01). Given that GYT088 exhibited dose- and time-dependent angiogenesis inhibition, it was determined that intravitreal injection of GYT088 (at doses of 2.56 and 5.12 ng / eye) was effective in inhibiting laser-induced angiogenesis. The equivalent human dose range is 2.56 and 5.12 μg / eye.

[0057] 4.2 Analysis of retinal sublayer structure

[0058] OCT images were used to observe the structural integrity of retinal sublayers and substructural changes. For example, some highly reflective nodes may represent immune cell aggregation or pigment epithelial cell migration. OCT images also revealed retinal cysts. To compare the severity of retinal damage observed in fundus photography among the groups, a score of 0 to 4 was assigned based on symptom severity. Results from three blinded groups were quantified and statistically analyzed (Figure 3). The results showed that on day 7, the damage scores in each group ranged from 0.80 to 1.62. The damage scores in the Eylea group (0.80±0.13) were significantly lower than those in the control group (1.38±0.17), demonstrating that Eylea protects retinal sublayers. On day 14, the damage scores in each group ranged from 0.93 to 2.62. The high-dose (5.12 ng / eye) GYT088 treatment group also showed a significant decrease in damage scores compared to the control group, demonstrating that GYT088 can maintain retinal sublayers and reduce cell aggregation.

[0059] While the present invention provides the preferred embodiments described above, they are not intended to limit the present invention. Any person skilled in the art may make modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be defined by the appended claims.

Claims

1. Use of a compound for preparing a drug for inhibiting angiogenesis, wherein the compound is a beauvericin having the following structure, and the therapeutically effective concentration range of the beauvericin is 0.1 μM to 5 μM:

2. The use according to claim 1, wherein the specific low dose range of sclerotin is 0.5 to 5 μM.

3. The use as claimed in claim 1, wherein the drug for inhibiting angiogenesis can be developed as a drug for treating or preventing retinopathy.

4. The use according to claim 3, wherein the retinopathy is selected from the group consisting of: age-related macular degeneration (AMD), glaucoma and diabetic retinopathy.

5. A pharmaceutical composition for inhibiting angiogenesis, comprising a therapeutically effective concentration of sclerotin in the range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier. 6 . The pharmaceutical composition of claim 5 , wherein the specific low dose range of sclerotin is 0.5 μM to 5 μM.

7. The pharmaceutical composition according to claim 5, which is a drug for treating or preventing retinopathy.

8. The pharmaceutical composition of claim 5, which can be combined with an anticancer drug to prepare a pharmaceutical composition or pharmaceutical combination for treating cancer.

Images